A self-powered submersible microbial electrolysis cell (SMEC), in which a specially designed anode chamber and external electricity supply were not needed, was developed for in situ biohydrogen production from anaerobic reactors. In batch experiments, the hydrogen production rate reached 17.8 mL/L/d at the initial acetate concentration of 410 mg/L (5 mM), while the cathodic hydrogen recovery (RH2) and overall systemic coulombic efficiency (CEos) were 93% and 28%, respectively, and the systemic hydrogen yield (YH2) peaked at 1.27 mol-H2/mol-acetate. The hydrogen production increased along with acetate and buffer concentration. The highest hydrogen production rate of 32.2 mL/L/d and YH2 of 1.43 mol-H2/mol-acetate were achieved at 1640 mg/L (20 mM) acetate and 100 mM phosphate buffer. Further evaluation of the reactor under single electricity-generating or hydrogen-producing mode indicated that further improvement of voltage output and reduction of electron losses were essential for efficient hydrogen generation. In addition, alternate exchanging the electricity-assisting and hydrogen-producing function between the two cell units of the SMEC was found to be an effective approach to inhibit methanogens. Furthermore, 16S rRNA genes analysis showed that this special operation strategy resulted same microbial community structures in the anodic biofilms of the two cell units. The simple, compact and in situ applicable SMEC offers new opportunities for reactor design for a microbial electricity-assisted biohydrogen production system.